The efficiency of stop codon recognition and the activation of downstream quality control depend on the RNA sequence, protein and spatial contexts of a particular stop codon. Genetic mutations and defects in messenger RNA (mRNA) processing often result in premature termination codons (PTCs). Translating these mRNAs produces truncated proteins that often have deleterious effects for the cell or organism. The effects of such mutations are buffered by nonsense-mediated mRNA decay (NMD), which selectively degrades mRNAs with PTCs, as well as hundreds of ?normal? mRNA species. Current models fail to explain how most PTCs are detected, particularly the molecular details that connect the mechanics of translation termination to mRNA decay. Stop codon readthrough, even at relatively low efficiency, can render a message resistant to NMD. There is also evidence that underlying mechanisms are shared between stop codon recognition and NMD. The goal of this proposal is to identify currently unknown factors involved in NMD and stop codon readthrough and to understand their biochemical roles in these processes, especially as it pertains to modulating the activity of ribosomes. In Aim 1 I will use CRISPR-Cas9 screening and mass spectrometry approaches to identify candidate protein factors that differentiate PTCs from normal stop codons and contribute to stop codon readthrough in vivo. The genes identified in these screens are potential targets for therapeutic intervention in genetic diseases caused by PTCs. In Aim 2, I will study the mechanism by which these factors cause stop codon read-through and NMD. I will use RNA-seq and enhanced ribosome footprint profiling to assess the generality of a factor's role in NMD or stop codon recognition in vivo and determine mechanistic details about its effect on stop codon recognition. Mechanistic hypotheses about a protein's effect on translation termination will be tested in a rabbit reticulocyte lysate translation system to determine how stop codon recognition is affected by these factors. These systems will enable structural characterization of the interaction of these factors with ribosomes or mRNA by high- throughput chemical probing and cryo-electron microscopy. Together, these aims will expand our understanding of the mechanistic underpinnings of premature stop codon recognition, and how this leads to mRNA decay or stop codon readthrough. The experiments I propose will advance my technical skills in ribosome biochemistry, human cell culture techniques, genome-wide CRISPR screening, and mass spectrometry. The latter three of these skillsets will be particularly important in the development of my own research program and its differentiation from that of my mentor. The advisory committee that I have assembled, and the accompanying training plan, will aid me in the development of other essential career skills, including presentation skills, paper and grant writing, and mentoring and leadership. The technical and career training I will receive will prepare me to start a tenure-track position as an independent scientist.